4,4'-[2,2,2-trifluoro-1-(trifluoromethyl)ethylidene]diphenol; bisphenol AF

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

endocrine disrupter mammalian screening – in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

The study was conducted to OECD 407

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other:

Version / remarks:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)

Deviations:

no

GLP compliance:

yes

Test type:

Repeated dose toxicity in rats

Limit test:

no

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Not reported
- Stability under test conditions: Not reported
- Solubility and stability of the test substance in the solvent/vehicle: Confirmed
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not reported

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Not reported
- Preliminary purification step (if any): Not reported
- Final dilution of a dissolved solid, stock liquid or gel: Not reported
- Final preparation of a solid: Not reported

FORM AS APPLIED IN THE TEST (if different from that of starting material): Not reported

OTHER SPECIFICS: n/a

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crj:CD(SD)

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Crj:CD (SD) rats were purchased from Charles River, Japan, Inc (Shiga, Japan)
- Females (if applicable) nulliparous and non-pregnant: Not reported
- Age at study initiation: 8 weeks
- Weight at study initiation: ca. 310 - 340 g (males) and ca. 203 - 226 g (females)
- Fasting period before study: Not reported
- Housing: Housed individually in stainless stell wire-mesh cages.
- Diet (e.g. ad libitum): Ad libitum, commercial diet (MF, Oriental Yeast Co., Tokyo, Japan)
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: Not reported

DETAILS OF FOOD AND WATER QUALITY: Not reported

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 ºC
- Humidity (%): 55 ± 20 %
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12: 12 (light: dark)

IN-LIFE DATES: Not reported

Route of administration:

oral: gavage

Details on route of administration:

The volume of the olive oil that contained the chemical for gavage was 5 mL/kg.

Vehicle:

olive oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): n/a
- Mixing appropriate amounts with (Type of food): n/a
- Storage temperature of food: n/a

VEHICLE
- Justification for use and choice of vehicle (if other than water): olive oil - OECD recommended to aid solubility of test item.
- Concentration in vehicle: Not reported
- Amount of vehicle (if gavage): 5 mL/kg/bw
- Lot/batch no. (if required): Not reported
- Purity: Not reported

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily

Dose / conc.:

10 mg/kg bw/day (nominal)

Dose / conc.:

30 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale: Chosen after completition of a range-finder test. Concentrations did not induce death or severe suffering.
- Rationale for animal assignment (if not random): Random
- Rationale for selecting satellite groups: No
- Post-exposure recovery period in satellite groups: n/a
- Section schedule rationale (if not random): Males were necropsied on Day 29. Females were necropsied after having been dosed for at least 29 days and killed ondays 30–33 to allow them to be killed in the diestrous stage or in the continuous same stage.

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- Cage side observations checked in Table 2 were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Twice weekly and before necropsy

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): n/a
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: n/a
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: n/a

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): n/a
- Time schedule for examinations: n/a

OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations: n/a
- Dose groups that were examined: n/a

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At termination
- Anaesthetic used for blood collection: Yes (chemical name not reported)
- Animals fasted: Not specified
- How many animals: 20 animals per treatment group (10 males and 10 females, 80 animals in total)
- Parameters checked in Table 3 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At termination
- Animals fasted: Not specified
- How many animals: 20 animals per treatment group (10 males and 10 females, 80 animals in total)
- Parameters checked in Table 3 were examined.

URINALYSIS: No
- Time schedule for collection of urine: n/a
- Metabolism cages used for collection of urine: n/a
- Animals fasted: n/a

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: 4th week after test initialtion
- Dose groups that were examined: Control, 10, 30 and 100 mg/kg bw/day
- Battery of functions tested: sensory activity / grip strength / motor activity.

IMMUNOLOGY: No
- Time schedule for examinations: n/a
- How many animals: n/a
- Dose groups that were examined: n/a

OTHER: Hormone analysis (in serum), estrous cycle, spermatology and histopathology observations were also undertaken.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes - Table 4

HISTOPATHOLOGY: Yes - Table 4

Statistics:

Bartlett’s variance test was performed for the parametric data (body weight, food consumption, hematological data, clinical biochemical data, hormonal data, organ weight, sperm count, grip strength, and locomotor activity). Bartlett’s test revealed a homogeneous variance, so one-way analysis of variance was conducted and if the result of the one-way analysis was significant, Dunnett’s test was performed to compare the treated and the control groups. Data with an inhomogeneous variance shown by Bartlett’s test or nonparametric data (the number of stools, the number of urinary pools, and incidence rate of abnormal spermatozoa) were subjected to the Kruskal–Wallis rank test, and if a significant divergence was observed, a Dunnett’s approach was carried out. Incidence rates of abnormal estrous cycles, gross pathological findings, and histopathological findings were analyzed by Fisher’s “exact” probability test. In the evaluation of the examination results, when a divergence from the control was found at a significance level of 1 or 5%, it was regarded as a significant change.

A male rat in the control group was diagnosed as being the subject of an administration error on gross and histopathological examination. The error was thought to have happened just before day 22, based on changes in body weights and food consumption, so data that contained body weights at days 26 and 28, food consumption at days 22 and 28, and all organ weights of this rat were excluded from the statistical analysis.

Endocrine disrupting potential:

positive

Maximum tolerated dose level exceeded:

not specified

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Several male and female rats in the 100 mg/kg group showed salivation from the first week, and this sign disappeared within 90 min of administration. No other abnormal general findings were observed in any of the groups.

Salivation in the 100 mg/kg group was not found to be a sign of toxicity because this sign disappeared soon after administration and no other related changes were observed.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

A decrease in body weight gains was found in the male rat 100 mg/kg group and the female rat 30 and 100 mg/kg groups from the first week after administration and was accompanied by decreased food consumption.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

A decrease in body weight gains was found in the male rat 100 mg/kg group and the female rat 30 and 100 mg/kg groups from the first week after administration and was accompanied by decreased food consumption.

Water consumption and compound intake (if drinking water study):

not examined

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In male rats, white blood cell counts, total cholesterol, and albumin values decreased in the 100 mg/kg group. In female rats, cholinesterase and total cholesterol values decreased and total bilirubin values increased in the 100 mg/kg group.

Serum T4 values increased in the 100 mg/kg groups of both sexes, but no changes in TSH were detected in any treated groups.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

In males, the relative weights of kidney, adrenal, and brain increased signifcantly in the 100 mg/kg group and the absolute weights of prostate, ventral prostate, seminal vesicle, liver, heart, and spleen decreased in this group. In female rats, the relative brain weights increased significantly in the 30 and 100 mg/kg groups, and the absolute heart weights decreased in the 100 mg/kg group.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Dilatation of the large intestinal lumen was observed in 9 male and female rats in the 100 mg/kg groups, respectively.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In the male rats, the incidence of changes, such as atrophy of testicular Leydig cells, hypertrophy of the adrenal zona fasciculata, and decreased hepatocytic glycogen, was higher in the 100 mg/kg group than in the control group. In addition, decreased hematopoiesis in the bone marrow and spleen, atrophy of the mammary glands, and atrophy of pituitary basophilic cells were also observed in the 100 mg/kg group. In female rats, hypertrophy of the adrenal zona fasciculata and decreased hepatocytic glycogen were detected in several rats given the chemical.

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 10 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

body weight

organ weights

gross pathology

histopathology

 

Table 5       Mean body weight changes following 28 days of treatment 

 

ose Group	Mean Initial Body Weight (g)		Mean Terminal Body Weight (g)
	Male	Female	Male	Female
Control	324 ± 14	211 ± 7	449 ± 27	274 ± 18
10 mg/kg/day	324 ± 13	215 ± 11	451 ± 26	277 ± 18
30 mg/kg/day	325 ± 15	213 ± 10	450 ± 33	255 ± 18*
100 mg/kg/day	326 ± 13	214 ± 11	396 ± 29**	253 ± 15*

* statistically different from control group (p < 0.05)

** statistically different from control group (p < 0.01)

 

Table 6:         Selected haematology, clinical chemistry and pathology findings 

 

aily Dose (mg/kg bw/day)	Male				Female
	Control	10	30	100	Control	10	30	100
No. of animals	10	10	10	10	10	10	10	10
Haematology
- WBC (x10^3/mm^3)	12.28 ± 1.05	11.83 ± 2.66	11.17 ± 2.67	9.17 ± 2.46*	8.59 ± 2.49	9.06 ± 2.15	9.28 ± 1.77	8.90 ± 1.84
Blood chemistry
- Cholinesterase (IU/L)	54 ±18	56 ± 19	48 ± 11	50 ± 10	525 ± 163	537 ± 98	465 ± 236	300 ± 127*
- Total cholesterol (mg/dL)	69 ± 8	60 ± 12	60 ± 9	49 ± 7**	69 ± 12	65 ± 7	60 ± 11	47 ± 5**
- Total bilirubin (mg/dL)	0.03 ± 0.02	0.02 ± 0.01	0.02 ± 0.01	0.03 ± 0.02	0.02 ± 0.01	0.01 ± 0.01	0.02 ± 0.01	0.04 ± 0.02**
- Albumin (g/dL)	3.00 ± 0.28	3.05 ± 0.12	3.05 ± 0.09	2.84 ± 0.11**	3.51 ± 0.45	3.56 ±0.16	3.35 ± 0.33	3.30 ± 0.27
- T4 (ng/dL)	3.704 ± 0.452	4.158 ± 0.826	4.061 ± 0.846	4.754 ± 0.762**	2.397 ± 0.297	2.376 ± 0.443	2.826 ± 0.950	3.671 ± 0.479**
Histopathology
- Bone marrow,decreased hematopoiesis	0	0	0	4	0	NE	NE	0
- Spleen,decreased extramedullary	0	0	0	2	0	NE	NE	0
- Testis,atrophy of Leydig cells	0	0	0	5*
- Mammary gland,atrophy of glands	0	0	0	3	0	NE	NE	0
- Adrenal gland,hypertrophy of zona fasiculata	1	1	0	8**	0	1	1	2
- Pituitary gland,atrophy of basophilic cells	0	0	0	1	0	NE	NE	0
- Liver,decreased hepatocytic glycogen	1	0	1	8**	0	0	1	2
- Organs in thoracic cavity,inflammation and granuloma	1	NE	NE	0	0	NE	NE	0

NE not examined

* significant difference from control (p < 0.05)

** significant difference from control (p <0.01)

 

Table 7:         Absolute and relative organ weights 

 

Daily Dose (mg/kg/day)	Male				Female
	Control	10	30	100	Control	10	30	100
No. of animals	9	10	10	10	10	10	10	10
Organ Weights
Prostate (ventral and dorsolateral)
mg	1068 ± 188	1134 ± 179	1061 ± 189	827 ± 183*	-	-	-	-
g/100 g	0.237 ± 0.045	0.251 ± 0.031	0.236 ± 0.041	0.208 ± 0.039	-	-	-	-
Ventral prostate
mg	631 ± 166	741 ± 110	676 ± 150	474 ± 112*	-	-	-	-
g/100 g	0.139 ± 0.034	0.164 ± 0.021	0.150 ± 0.030	0.120 ± 0.027	-	-	-	-
Seminal vesicle
g	1.41 ± 0.19	1.43 ± 0.30	1.38 ± 0.24	1.02 ± 0.36*	-	-	-	-
g/100 g	0.312 ± 0.045	0.317 ± 0.069	0.307 ± 0.045	0.254 ± 0.079	-	-	-	-
Liver
g	17.13 ± 2.18	16.90 ± 1.37	16.38 ± 2.85	14.10 ± 1.31**	-	-	-	-
g/100 g	3.778 ± 0.322	3.747 ± 0.179	3.623 ± 0.399	3.564 ± 0.271	-	-	-	-
Kidney
g	3.02 ± 0.23	3.01 ± 0.18	3.00 ± 0.37	2.89 ± 0.30	-	-	-	-
g/100 g	0.667 ± 0.021	0.671 ± 0.058	0.666 ± 0.064	0.729 ± 0.043*	-	-	-	-
Heart
g	1.28 ± 0.09	1.32 ± 0.08	1.28 ± 0.09	1.13 ± 0.13**	0.87 ± 0.08	0.88 ± 0.05	0.82 ± 0.11	0.78 ± 0.06*
g/100 g	0.283 ± 0.013	0.293 ± 0.018	0.285 ± 0.011	0.286 ± 0.017	0.316 ± 0.015	0.319 ± 0.021	0.321 ± 0.024	0.308 ± 0.020
Spleen
g	0.71 ± 0.08	0.70 ± 0.08	0.71 ± 0.10	0.59 ± 0.09*	-	-	-	-
g/100 g	0.158 ± 0.018	0.155 ± 0.015	0.158 ± 0.020	0.149 ± 0.016	-	-	-	-
Adrenals
mg	58 ± 8	58 ± 11	56 ± 4	63 ± 9	-	-	-	-
g/100 g	0.013 ± 0.002	0.013 ± 0.002	0.012 ± 0.001	0.016 ± 0.003**	-	-	-	-
Brain
g	2.17 ± 0.05	2.21 ± 0.09	2.23 ± 0.06	2.19 ± 0.07	2.03 ± 0.05	2.00 ± 0.06	2.05 ± 0.10	2.02 ± 0.08
g/100 g	0.482 ± 0.034	0491 ± 0.032	0.498 ± 0.036	0.554 ± 0.035**	0.742 ± 0.042	0.725 ± 0.044	0.809 ± 0.070*	0.799 ± 0.039*

* significantly different from control (p < 0.05)

** significantly different from control (p < 0.01)

 

Conclusions:

The no adverse effect level (NOAEL) for the test item was determined to be 10 mg/kg bw/day, due to findings of reduced body weight gain and abnormal estrous cycles in the female rat 30 mg/kg bw/day group.

Executive summary:

In a subacute repeat dose toxicity study (OECD 407), Bisphenol AF was administered to 30 male and 30 female Sprague-Dawley rats by oral gavage at dose levels of 10, 30 and 100 mg/kg bw/day for up to 28 consecutive days. In addition, control rats were dosed with vehicle (olive oil) alone for each test group.

A summary of adult responses to Bisphenol AF is described below;

Mortality - no treatment-related deaths were detected.

Clinical signs - clinical signs were generally confined to post-dose increased salvation and staining around the mouth for animals treated at 100 mg/kg/day. Regression of all signs was evident 90 minutes after treatment.

Behavioural assessments - open field observations did not reveal any treatment-related effects.

Functional performance - no treatment-related effects were evident for grip or motor activity.

Sensory reactivity assessments - no treatment-related effects were observed.

Bodyweight - significant (p< 0.01) reduced bodyweight gains were evident for males in the 100 mg/kg/day group during the treatment period. For females, bodyweight gain was significantly (p <0.05) reduced in the 30 and 100 mg/kg/day groups.

Haematology and blood chemistry - for males, a significant reduction in white blood cell count (p < 0.05), cholinesterase and albumin (p < 0.01) was observed in the 100 mg/kg bw/day treatment group. Further, a significant (p < 0.01) increase in T4 hormone was also observed in the same treatment group. For females, a significant decrease in cholinesterase (p < 0.05) and total cholesterol (p < 0.01) was observed in the 100 mg/kg bw/day treatment group. Further, a significant (p < 0.01) increase in total bilirubin and T4 hormone was also observed in the same treatment group.

Oestrous cycle assessment - irregular estrous cycles were observed in the 30 and 100 mg/kg groups, and the diestrous stage continued in some animals. The mean duration of estrous cycles was prolonged in the 100 mg/kg group without statistical difference.

Necropsy - dilatation of the large intestinal lumen was observed in 9 male and female rats in the 100 mg/kg groups, respectively.

Organ weights - in males, the relative weights of kidney, adrenal, and brain increased significantly in the 100 mg/kg group and the absolute weights of prostate, ventral prostate, seminal vesicle, liver, heart, and spleen decreased in the same group. In female rats, the relative brain weights increased significantly in the 30 and 100 mg/kg groups, and the absolute heart weights decreased in the 100 mg/kg group.

Histopathological changes - in the male rats, the incidence of changes, such as atrophy of testicular Leydig cells, hypertrophy of the adrenal zona fasciculata, and decreased hepatocytic glycogen, was statistically significantly higher in the 100 mg/kg group than in the control group. In addition, decreased hematopoiesis in the bone marrow and spleen, atrophy of the mammary glands, and atrophy of pituitary basophilic cells were also observed in the 100 mg/kg group. In female rats, hypertrophy of the adrenal zona fasciculata and decreased hepatocytic glycogen were detected in several rats at each dose level, although these changes were not statistically significantly.

In conclusion, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity was 10 mg/kg bw/day due to findings of reduced body weight gain and abnormal estrous cycles in the female rat treated at 30 mg/kg bw/day.

This subacute repeat dose toxicity study in the rat is acceptable and satisfies the guideline requirements for an OECD 407 in the rat.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2012

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4,4'-[2,2,2-trifluoro-1-(trifluoromethyl)ethylidene]diphenol

Cas Number:

1478-61-1

Molecular formula:

C15H10F6O2

IUPAC Name:

4,4'-[2,2,2-trifluoro-1-(trifluoromethyl)ethylidene]diphenol

Test material form:

not specified

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Not reported
- Stability under test conditions: Not reported
- Solubility and stability of the test substance in the solvent/vehicle: Confirmed
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not reported

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Not reported
- Preliminary purification step (if any): Not reported
- Final dilution of a dissolved solid, stock liquid or gel: Not reported
- Final preparation of a solid: Not reported

FORM AS APPLIED IN THE TEST (if different from that of starting material): Not reported

OTHER SPECIFICS: n/a

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Details on species / strain selection:

Crj:CD (SD) rats were purchased from Charles River, Japan, Inc (Shiga, Japan)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Crj:CD (SD) rats were purchased from Charles River, Japan, Inc (Shiga, Japan)
- Females (if applicable) nulliparous and non-pregnant: Not reported
- Age at study initiation: 8 weeks
- Weight at study initiation: ca. 310 - 340 g (males) and ca. 203 - 226 g (females)
- Fasting period before study: Not reported
- Housing: Housed individually in stainless stell wire-mesh cages.
- Diet (e.g. ad libitum): Ad libitum, commercial diet (MF, Oriental Yeast Co., Tokyo, Japan)
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: Not reported

DETAILS OF FOOD AND WATER QUALITY: Not reported

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 ºC
- Humidity (%): 55 ± 20 %
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12: 12 (light: dark)

IN-LIFE DATES: Not reported

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The volume of the olive oil that contained the chemical for gavage was 5 mL/kg.

Vehicle:

olive oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): n/a
- Mixing appropriate amounts with (Type of food): n/a
- Storage temperature of food: n/a

VEHICLE
- Justification for use and choice of vehicle (if other than water): olive oil - OECD recommended to aid solubility of test item.
- Concentration in vehicle: Not reported
- Amount of vehicle (if gavage): 5 mL/kg/bw
- Lot/batch no. (if required): Not reported
- Purity: Not reported

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

10 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

30 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Chosen after completition of a range-finder test. Concentrations did not induce death or severe suffering.
- Rationale for animal assignment (if not random): Random
- Rationale for selecting satellite groups: No
- Post-exposure recovery period in satellite groups: n/a
- Section schedule rationale (if not random): Males were necropsied on Day 29. Females were necropsied after having been dosed for at least 29 days and killed ondays 30–33 to allow them to be killed in the diestrous stage or in the continuous same stage.

Positive control:

No

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- Cage side observations checked in Table 2 were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Twice weekly and before necropsy

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): n/a
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: n/a
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: n/a

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): n/a
- Time schedule for examinations: n/a

OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations: n/a
- Dose groups that were examined: n/a

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At termination
- Anaesthetic used for blood collection: Yes (chemical name not reported)
- Animals fasted: Not specified
- How many animals: 20 animals per treatment group (10 males and 10 females, 80 animals in total)
- Parameters checked in Table 3 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At termination
- Animals fasted: Not specified
- How many animals: 20 animals per treatment group (10 males and 10 females, 80 animals in total)
- Parameters checked in Table 3 were examined.

URINALYSIS: No
- Time schedule for collection of urine: n/a
- Metabolism cages used for collection of urine: n/a
- Animals fasted: n/a

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: 4th week after test initialtion
- Dose groups that were examined: Control, 10, 30 and 100 mg/kg bw/day
- Battery of functions tested: sensory activity / grip strength / motor activity.

IMMUNOLOGY: No
- Time schedule for examinations: n/a
- How many animals: n/a
- Dose groups that were examined: n/a

OTHER: Hormone analysis (in serum), estrous cycle, spermatology and histopathology observations were also undertaken.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes - Table 4

HISTOPATHOLOGY: Yes - Table 4

Statistics:

Bartlett’s variance test was performed for the parametric data (body weight, food consumption, hematological data, clinical biochemical data, hormonal data, organ weight, sperm count, grip strength, and locomotor activity). Bartlett’s test revealed a homogeneous variance, so one-way analysis of variance was conducted and if the result of the one-way analysis was significant, Dunnett’s test was performed to compare the treated and the control groups. Data with an inhomogeneous variance shown by Bartlett’s test or nonparametric data (the number of stools, the number of urinary pools, and incidence rate of abnormal spermatozoa) were subjected to the Kruskal–Wallis rank test, and if a significant divergence was observed, a Dunnett’s approach was carried out. Incidence rates of abnormal estrous cycles, gross pathological findings, and histopathological findings were analyzed by Fisher’s “exact” probability test. In the evaluation of the examination results, when a divergence from the control was found at a significance level of 1 or 5%, it was regarded as a significant change.

A male rat in the control group was diagnosed as being the subject of an administration error on gross and histopathological examination. The error was thought to have happened just before day 22, based on changes in body weights and food consumption, so data that contained body weights at days 26 and 28, food consumption at days 22 and 28, and all organ weights of this rat were excluded from the statistical analysis.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Several male and female rats in the 100 mg/kg group showed salivation from the first week, and this sign disappeared within 90 min of administration. No other abnormal general findings were observed in any of the groups.

Salivation in the 100 mg/kg group was not found to be a sign of toxicity because this sign disappeared soon after administration and no other related changes were observed.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

A decrease in body weight gains was found in the male rat 100 mg/kg group and the female rat 30 and 100 mg/kg groups from the first week after administration and was accompanied by decreased food consumption.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

A decrease in body weight gains was found in the male rat 100 mg/kg group and the female rat 30 and 100 mg/kg groups from the first week after administration and was accompanied by decreased food consumption.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

In male rats, white blood cell counts, total cholesterol, and albumin values decreased in the 100 mg/kg group. In female rats, cholinesterase and total cholesterol values decreased and total bilirubin values increased in the 100 mg/kg group.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In male rats, white blood cell counts, total cholesterol, and albumin values decreased in the 100 mg/kg group. In female rats, cholinesterase and total cholesterol values decreased and total bilirubin values increased in the 100 mg/kg group.

Serum T4 values increased in the 100 mg/kg groups of both sexes, but no changes in TSH were detected in any treated groups.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

In males, the relative weights of kidney, adrenal, and brain increased signifcantly in the 100 mg/kg group and the absolute weights of prostate, ventral prostate, seminal vesicle, liver, heart, and spleen decreased in this group. In female rats, the relative brain weights increased significantly in the 30 and 100 mg/kg groups, and the absolute heart weights decreased in the 100 mg/kg group.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Dilatation of the large intestinal lumen was observed in 9 male and female rats in the 100 mg/kg groups, respectively.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In the male rats, the incidence of changes, such as atrophy of testicular Leydig cells, hypertrophy of the adrenal zona fasciculata, and decreased hepatocytic glycogen, was higher in the 100 mg/kg group than in the control group. In addition, decreased hematopoiesis in the bone marrow and spleen, atrophy of the mammary glands, and atrophy of pituitary basophilic cells were also observed in the 100 mg/kg group. In female rats, hypertrophy of the adrenal zona fasciculata and decreased hepatocytic glycogen were detected in several rats given the chemical.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Sperm analysis - no effects observed.

Estrous cycle - effects observed, treatment-related. Irregular estrous cycles were observed in the 30 and 100 mg/kg groups, and the diestrous stage continued in some animals. The mean duration of estrous cycles was prolonged in the 100 mg/kg group without statistical diverence (0 mg/kg group = 4.2 ± 0.4 days, 100 mg/kg group = 4.9 ± 0.9 days), and estrous cycling days were not measured in one and three of 10 rats in the 30 and 100 mg/kg groups, respectively, due to the irregularity of their estrous cycles.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Remarks on result:

other:

Target system / organ toxicity

1

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day (nominal)

System:

male reproductive system

Organ:

seminal vesicle

other: Prostate weights

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

2

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day (nominal)

System:

endocrine system

Organ:

adrenal glands

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Any other information on results incl. tables

Table 5       Mean body weight changes following 28 days of treatment

Dose Group	Mean Initial Body Weight (g)		Mean Terminal Body Weight (g)
	Male	Female	Male	Female
Control	324 ± 14	211 ± 7	449 ± 27	274 ± 18
10 mg/kg/day	324 ± 13	215 ± 11	451 ± 26	277 ± 18
30 mg/kg/day	325 ± 15	213 ± 10	450 ± 33	255 ± 18*
100 mg/kg/day	326 ± 13	214 ± 11	396 ± 29**	253 ± 15*

* statistically different from control group (p < 0.05)

** statistically different from control group (p < 0.01)

Table 6:         Selected haematology, clinical chemistry and pathology findings

 

Daily Dose (mg/kg bw/day)	Male				Female
	Control	10	30	100	Control	10	30	100
No. of animals	10	10	10	10	10	10	10	10
Haematology
- WBC (x10^3/mm^3)	12.28 ± 1.05	11.83 ± 2.66	11.17 ± 2.67	9.17 ± 2.46*	8.59 ± 2.49	9.06 ± 2.15	9.28 ± 1.77	8.90 ± 1.84
Blood chemistry
- Cholinesterase (IU/L)	54 ±18	56 ± 19	48 ± 11	50 ± 10	525 ± 163	537 ± 98	465 ± 236	300 ± 127*
- Total cholesterol (mg/dL)	69 ± 8	60 ± 12	60 ± 9	49 ± 7**	69 ± 12	65 ± 7	60 ± 11	47 ± 5**
- Total bilirubin (mg/dL)	0.03 ± 0.02	0.02 ± 0.01	0.02 ± 0.01	0.03 ± 0.02	0.02 ± 0.01	0.01 ± 0.01	0.02 ± 0.01	0.04 ± 0.02**
- Albumin (g/dL)	3.00 ± 0.28	3.05 ± 0.12	3.05 ± 0.09	2.84 ± 0.11**	3.51 ± 0.45	3.56 ±0.16	3.35 ± 0.33	3.30 ± 0.27
- T4 (ng/dL)	3.704 ± 0.452	4.158 ± 0.826	4.061 ± 0.846	4.754 ± 0.762**	2.397 ± 0.297	2.376 ± 0.443	2.826 ± 0.950	3.671 ± 0.479**
Histopathology
- Bone marrow,decreased hematopoiesis	0	0	0	4	0	NE	NE	0
- Spleen,decreased extramedullary	0	0	0	2	0	NE	NE	0
- Testis,atrophy of Leydig cells	0	0	0	5*
- Mammary gland,atrophy of glands	0	0	0	3	0	NE	NE	0
- Adrenal gland,hypertrophy of zona fasiculata	1	1	0	8**	0	1	1	2
- Pituitary gland,atrophy of basophilic cells	0	0	0	1	0	NE	NE	0
- Liver,decreased hepatocytic glycogen	1	0	1	8**	0	0	1	2
- Organs in thoracic cavity,inflammation and granuloma	1	NE	NE	0	0	NE	NE	0

NE not examined

* significant difference from control (p < 0.05)

** significant difference from control (p <0.01)

Table 7:         Absolute and relative organ weights

 

Daily Dose (mg/kg/day)	Male				Female
	Control	10	30	100	Control	10	30	100
No. of animals	9	10	10	10	10	10	10	10
Organ Weights
Prostate (ventral and dorsolateral)
mg	1068 ± 188	1134 ± 179	1061 ± 189	827 ± 183*	-	-	-	-
g/100 g	0.237 ± 0.045	0.251 ± 0.031	0.236 ± 0.041	0.208 ± 0.039	-	-	-	-
Ventral prostate
mg	631 ± 166	741 ± 110	676 ± 150	474 ± 112*	-	-	-	-
g/100 g	0.139 ± 0.034	0.164 ± 0.021	0.150 ± 0.030	0.120 ± 0.027	-	-	-	-
Seminal vesicle
g	1.41 ± 0.19	1.43 ± 0.30	1.38 ± 0.24	1.02 ± 0.36*	-	-	-	-
g/100 g	0.312 ± 0.045	0.317 ± 0.069	0.307 ± 0.045	0.254 ± 0.079	-	-	-	-
Liver
g	17.13 ± 2.18	16.90 ± 1.37	16.38 ± 2.85	14.10 ± 1.31**	-	-	-	-
g/100 g	3.778 ± 0.322	3.747 ± 0.179	3.623 ± 0.399	3.564 ± 0.271	-	-	-	-
Kidney
g	3.02 ± 0.23	3.01 ± 0.18	3.00 ± 0.37	2.89 ± 0.30	-	-	-	-
g/100 g	0.667 ± 0.021	0.671 ± 0.058	0.666 ± 0.064	0.729 ± 0.043*	-	-	-	-
Heart
g	1.28 ± 0.09	1.32 ± 0.08	1.28 ± 0.09	1.13 ± 0.13**	0.87 ± 0.08	0.88 ± 0.05	0.82 ± 0.11	0.78 ± 0.06*
g/100 g	0.283 ± 0.013	0.293 ± 0.018	0.285 ± 0.011	0.286 ± 0.017	0.316 ± 0.015	0.319 ± 0.021	0.321 ± 0.024	0.308 ± 0.020
Spleen
g	0.71 ± 0.08	0.70 ± 0.08	0.71 ± 0.10	0.59 ± 0.09*	-	-	-	-
g/100 g	0.158 ± 0.018	0.155 ± 0.015	0.158 ± 0.020	0.149 ± 0.016	-	-	-	-
Adrenals
mg	58 ± 8	58 ± 11	56 ± 4	63 ± 9	-	-	-	-
g/100 g	0.013 ± 0.002	0.013 ± 0.002	0.012 ± 0.001	0.016 ± 0.003**	-	-	-	-
Brain
g	2.17 ± 0.05	2.21 ± 0.09	2.23 ± 0.06	2.19 ± 0.07	2.03 ± 0.05	2.00 ± 0.06	2.05 ± 0.10	2.02 ± 0.08
g/100 g	0.482 ± 0.034	0491 ± 0.032	0.498 ± 0.036	0.554 ± 0.035**	0.742 ± 0.042	0.725 ± 0.044	0.809 ± 0.070*	0.799 ± 0.039*

* significantly different from control (p < 0.05)

** significantly different from control (p < 0.01)

Applicant's summary and conclusion

Conclusions:

The no adverse effect level (NOAEL) for the test item was determined to be 10 mg/kg bw/day, due to findings of reduced body weight gain and abnormal estrous cycles in the female rat 30 mg/kg bw/day group.

Executive summary:

In a subacute repeat dose toxicity study (OECD 407), Bisphenol AF was administered to 30 male and 30 female Sprague-Dawley rats by oral gavage at dose levels of 10, 30 and 100 mg/kg bw/day for up to 28 consecutive days. In addition, control rats were dosed with vehicle (olive oil) alone for each test group.

A summary of adult responses to Bisphenol AF is described below;

Mortality - no treatment-related deaths were detected.

Clinical signs - clinical signs were generally confined to post-dose increased salvation and staining around the mouth for animals treated at 100 mg/kg/day. Regression of all signs was evident 90 minutes after treatment.

Behavioural assessments - open field observations did not reveal any treatment-related effects.

Functional performance - no treatment-related effects were evident for grip or motor activity.

Sensory reactivity assessments - no treatment-related effects were observed.

Bodyweight - significant (p< 0.01) reduced bodyweight gains were evident for males in the 100 mg/kg/day group during the treatment period. For females, bodyweight gain was significantly (p <0.05) reduced in the 30 and 100 mg/kg/day groups.

Haematology and blood chemistry - for males, a significant reduction in white blood cell count (p < 0.05), cholinesterase and albumin (p < 0.01) was observed in the 100 mg/kg bw/day treatment group. Further, a significant (p < 0.01) increase in T4 hormone was also observed in the same treatment group. For females, a significant decrease in cholinesterase (p < 0.05) and total cholesterol (p < 0.01) was observed in the 100 mg/kg bw/day treatment group. Further, a significant (p < 0.01) increase in total bilirubin and T4 hormone was also observed in the same treatment group.

Oestrous cycle assessment - irregular estrous cycles were observed in the 30 and 100 mg/kg groups, and the diestrous stage continued in some animals. The mean duration of estrous cycles was prolonged in the 100 mg/kg group without statistical difference.

Necropsy - dilatation of the large intestinal lumen was observed in 9 male and female rats in the 100 mg/kg groups, respectively.

Organ weights - in males, the relative weights of kidney, adrenal, and brain increased significantly in the 100 mg/kg group and the absolute weights of prostate, ventral prostate, seminal vesicle, liver, heart, and spleen decreased in the same group. In female rats, the relative brain weights increased significantly in the 30 and 100 mg/kg groups, and the absolute heart weights decreased in the 100 mg/kg group.

Histopathological changes - in the male rats, the incidence of changes, such as atrophy of testicular Leydig cells, hypertrophy of the adrenal zona fasciculata, and decreased hepatocytic glycogen, was statistically significantly higher in the 100 mg/kg group than in the control group. In addition, decreased hematopoiesis in the bone marrow and spleen, atrophy of the mammary glands, and atrophy of pituitary basophilic cells were also observed in the 100 mg/kg group. In female rats, hypertrophy of the adrenal zona fasciculata and decreased hepatocytic glycogen were detected in several rats at each dose level, although these changes were not statistically significantly.

In conclusion, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity was 10 mg/kg bw/day due to findings of reduced body weight gain and abnormal estrous cycles in the female rat treated at 30 mg/kg bw/day.

This subacute repeat dose toxicity study in the rat is acceptable and satisfies the guideline requirements for an OECD 417 in the rat.